CN101706498B - Preparing method of electrochemiluminescence immunosensor - Google Patents
Preparing method of electrochemiluminescence immunosensor Download PDFInfo
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- CN101706498B CN101706498B CN 200910212772 CN200910212772A CN101706498B CN 101706498 B CN101706498 B CN 101706498B CN 200910212772 CN200910212772 CN 200910212772 CN 200910212772 A CN200910212772 A CN 200910212772A CN 101706498 B CN101706498 B CN 101706498B
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Abstract
The invention relates to a preparing technology of a nano immunological marker, in particular to a preparing method of an electrochemiluminescence immunosensor which has signal amplification function, is decorated by both SiO2 package luminescent material (Ru (bpy) 32 +) and a second antibody (Ab2) and used for low-concentration antigen detection. SiO2 core-shell type nano structure can not only well maintain the chemical property of the content, but also effectively prevent the leakage of the content. Since a plurality of marker molecules can be packaged in an SiO2 bead, the detection sensitivity is greatly improved. The grain diameter of synthesized SiO2@Ru is uniform and the SiO2@Ru has good monodispersity, therefore, identical amount of alpha-fetoprotein antibodies are fixed for each SiO2@Ru nano bead, and the detection repeatability is improved. The linear relation between ECL signal value and AFP concentration is good within the range of 0.01-20ng MmL-1, and the detection lower limit reaches 35pg mL-1.
Description
Technical field
The present invention relates to a kind of technology of preparing of nano immunity marker, related to have signal amplifying function by SiO
2Parcel luminescent substance (Ru (bpy)
3 2+) and the co-modified preparation method who is used for the electrochemiluminescimmunosensor immunosensor that low concentration antigen detects of second antibody (Ab2).
Background technology
Primary carcinoma of liver (hepatocellular carcinoma, the HCC) incidence of disease in recent years is gradually ascendant trend.HCC poor prognosis, 5 years survival rates are less than 5%, and the whole world has 5,918 ten thousand people to die from HCC every year approximately.From the nineties in 20th century, because the HCC death toll ranks the 3rd in world's mortality of malignant tumors number, China HCC is the cancer " killer " that comes second, and annual death toll accounts for the over half of world HCC death toll.One of reason that prognosis in hcc is poor is that most of patients is failed early detection and early diagnosis, and HCC result for the treatment of and the prognosis of different phase have notable difference.Therefore, early detection, early diagnosis and early treatment are the key factors that improves HCC survival of patients and prognosis.
In cancer patient's therapeutic process, the antigen that serum tumor is relevant can be used for the recurrence that noninvasive diagnostic test detects cancer, and behind patient undergoing treatment, its detection level plays an important role to patient's monitoring.Alpha-fetoprotein (alpha-fetoprotein, AFP) is present clinical unique widely used HCC diagnostic serum label, and AFP is used for generaI investigation and the examination of liver cancer high risk population, makes the shorter survival of some patients were and prognosis that significant raising arranged.α-fetoprotein (AFP) is the cancer protein that a kind of molecular weight is about 70kDa.Usually in the growth course of fetus an d neonate, drained by liver, yolk bag and alimentary canal.AFP is in the one-tenth human body of health, and its value is lower than 25ng/mL.The increase of AFP level is regarded as the early indication of some cancerations in the serum, and comprising liver cancer, yolk sac tumor is by cancer of the stomach, carcinoma of testis and the nasopharyngeal carcinoma of hepatic tissue transfer.At present, detect (ELISA) by Enzyme-linked Immunosorbent Assay, surface plasma body resonant vibration, fluorescence immunoassay, chemiluminescence, the detection meanss such as atomic absorption spectrum have had certain development in the detection of AFP antigen.
Detection method is highly sensitive because of it, selectivity well is subject to people in the analysis science field and pays close attention to widely for electrogenerated chemiluminescence (Electrochemiluminescence is called for short ECL).Electrochemiluminescence is to apply certain voltage at electrode, the chemiluminescence phenomenon of utilizing electrochemical reaction to cause directly or indirectly, and it is actually a chemiluminescent branch.The discovery of electrochemiluminescence can be traced back at the beginning of last century, but the application and research in the analytical chemistry field starts from 20 century 70s, and particularly after the eighties in 20th century, electrochemiluminescence has obtained flourish.At present correlative study has caused the great interest bipyridyl ruthenium of people (Ru (bpy)
3 2+) owing to have good water solubility, stable chemical performance, redox reversible, luminescence efficiency is high, the pH wide ranges of application, but characteristics such as electrochemical regeneration and lifetime of excited state be long and be widely used in the research of ECL.
In the research of electrochemiluminescence, the reagent that the method by chemical modification will directly or indirectly participate in chemiluminescence reaction is fixed on a class experimental provision that makes up on the electrode and is loosely referred to as electrochemical luminous sensor.In some cases, Highgrade integration and microminiaturized electrochemiluminescence device are also referred to as sensor sometimes.Because electrochemical luminous sensor has reduced the use of valuable reagent to a certain extent, and experimental provision is oversimplified or microminiaturization, day by day come into one's own in recent years.Ru (bpy)
3 2+Process for fixation roughly can be divided into the Nafion embrane method, LB embrane method, molecular self-assembling embrane method and sol-gel process.The method ubiquity poor stability of existing fixedly bipyridyl ruthenium, serviceable life is short, the problems such as the easy leakage of trim.Researching and developing new immobilization material will be the emphasis of Future Development.
Summary of the invention
Technical matters: the objective of the invention is for above technical matters, provide a kind of preparation technology simple, reduce the solid-state ECL immunosensor of testing cost, by the SiO to making
2The Ru (bpy) of parcel
3 2+Nanometer bead (SiO
2@Ru) surface-functionalized, obtain can be used for the low concentration biomolecule detection and having the composite nanoparticle label of signal amplification by the AFP antibody modification; Electricity consumption causes the antigen in the chemiluminescence method detection serum.
Technical scheme: a kind of preparation method of electrochemiluminescimmunosensor immunosensor, preparation process is: SiO
2The preparation of@Ru microballoon: with the 7.5mL cyclohexane, 1.8mL n-hexyl alcohol and 1.77mL Tween-80 mix, and then add 340 μ L 5mg/mL Ru (bpy)
3 2+Aqueous solution, uniform stirring reaction 30min forms stable Water-In-Oil system under the room temperature, again adding 100 μ L TEOS and 60 μ L NH in this system
3H
2O comes initiated polymerization, continue stirring reaction 20h under the room temperature, after reaction is finished, adding 5mL acetone in the system makes nano particle precipitation and separation is out from water in oil system, then with the centrifugal 10min of 8000r/min, can see having orange-yellow precipitation to form abandoning supernatant in the centrifuge tube bottom, with absolute ethyl alcohol and water difference washing precipitation several, fully remove the Ru (bpy) of surfactant TEOS and nano grain surface absorption
3 2+, get SiO
2@Ru microballoon; The preparation of nano immunity marker: AFP antibody modification SiO
2@Ru microballoon: 2mL SiO
2@Ru is diluted to 6mL with ethanol, adds 400 μ L APTS stirring reaction 30min, and is centrifugal, and water and ethanol washing obtain the SiO that APTS modifies for several times to remove unnecessary APTS
2@Ru; SiO with the APTS modification
2The glutaraldehyde solution of@Ru nano particle and 5mL 5wt% is at 37 ℃ of stirred in water bath reaction 2h, and is centrifugal and be scattered in the 3mL distilled water, and adds 2mL anti-AFP (Ab2), again at 37 ℃ of stirred in water bath reaction 2h, the centrifugal Ab2-SiO that obtains
2@Ru; At last, with Ab2-SiO
2@Ru is scattered in 5mL 1wt%BSA solution and stirs 30min, seals unnecessary amino, and is centrifugal, and with Ab2-SiO
2@Ru is dispersed to 2mL 0.02M pH 7.0PBS, and 4 ℃ save backup, and can obtain electrochemiluminescimmunosensor immunosensor thus.
The SiO that the reverse microemulsion method is synthetic
2The Ru (bpy) of parcel
3 2+Nanometer bead (SiO
2@Ru), can be used as the label of model protein alpha-fetoprotein antibody (anti-AFP), realized sensitive determination to alpha-fetoprotein (AFP) with the ECL method.SiO
2The nanostructured of core-shell type not only can well keep the chemical property of content, can also effectively stop the leakage of content.And because SiO
2Can wrap up a plurality of marker molecules in the bead, thereby the sensitivity of detection is greatly improved.
The specific surface of nano-silicon dioxide particle is large, and there is abundant hydroxyl on the surface.Therefore, by with APTES (APTS) reaction, just can be in its finishing under the room temperature amino group.End group is-NH
2Silicon dioxide granule is fixed on SiO take glutaraldehyde (glutaraldehyde) as crosslinking chemical with two anti-(Ab2)
2@Ru microsphere surface.This kind label can be used as the electrogenerated chemiluminescence signal tracer in the serum antigen sandwich immunoassay testing process, utilizes the electrogenerated chemiluminescence method to carry out immune detection, because a SiO
2Can wrap up a plurality of marker molecules in the bead, improve greatly the sensitivity that detects.
Beneficial effect: the SiO that (1) the present invention synthesizes with the reverse microemulsion method
2The Ru (bpy) of parcel
3 2+Nanometer bead (SiO
2@Ru), uniform particle diameter has good dispersiveness.SiO
2The nanostructured of core-shell type not only can well keep the chemical property of content, can also effectively stop the leakage of content.The ratio of the ratio of the particle diameter of nano particle and water and TEOS and water and surfactant Tween-80 has much relations, with TEOS: NH
3H
2O=10: 6 (volume ratios), H
2O: Tween-80=10: 52 (volume ratios), with the standby SiO of reverse microemulsion legal system
2The size homogeneous of@Ru nano particle, measuring its mean grain size by TEM is 140 ± 5nm.
(2) SiO
2@Ru can be used as the label of model protein alpha-fetoprotein antibody (anti-AFP), has realized sensitive determination to alpha-fetoprotein (AFP) with the ECL method.And because SiO
2Can wrap up a plurality of marker molecules in the bead, thereby the sensitivity of detection is greatly improved.
(3) with the SiO of uniform particle diameter
2@Ru microballoon is as modified antibodies and carrier, on the basis of having improved the sensitivity that detects, so that the immunosensor that makes has good repeatability and stability.
(4) technology of preparing of nano immunity marker is with SiO
2@Ru microballoon and APTS reaction, thus on microsphere surface the grafting amino group, utilize amino group that microballoon covers and the amino in the antibody at the cross-linking reaction realization antibody of glutaraldehyde at SiO
2Fixing of@Ru microsphere surface, then, with the SiO after 1% bovine serum albumin(BSA) (BSA) the processing modification
2@Ru microballoon, residual active epoxy group and the nonspecific binding site of sealing microsphere surface obtains a plurality of Ru (bpy)
3 2+The second antibody nano immune mark of molecular labeling.
(5) by using this label, but and can reach 0.035ng mL in conjunction with the low concentration biomolecule detection detectable antigens Cmin of Sandwich immunoassay
-1, the range of linearity is 0.05～20ng mL
-1, can verify that by the antigen of measuring a series of variable concentrations this detection method linearly dependent coefficient reaches 0.9986.
(6) SiO
2The nanostructured of core-shell type not only can well keep the chemical property of content, can also effectively stop the leakage of content.And because SiO
2Can wrap up a plurality of marker molecules in the bead, thereby the sensitivity of detection is greatly improved.
Description of drawings
Fig. 1 is that particle diameter is at the SiO of 140 ± 5nm
2@Ru bead;
Fig. 2 is SiO
2(a), SiO
2@Ru (b), free state Ru (bpy)
3 2+(c) PL spectrogram;
Fig. 3 is the synthetic synoptic diagram of nano immune mark;
Fig. 4 is sandwich immunoassay process synoptic diagram;
Fig. 5 is the resulting ECL signals of different AFP concentration; (a) 0.01, (b) 0.1, (c) 3, (d) 5, (e) 10, (f) 20ng mL
-1
Fig. 6 is the linear relationship of AFP concentration and ECL signal.
Embodiment
Embodiment 1:
A kind of preparation method of electrochemiluminescimmunosensor immunosensor, preparation process is: SiO
2The preparation of@Ru microballoon: with the 7.5mL cyclohexane, 1.8mL n-hexyl alcohol and 1.77mL Tween-80 mix, and then add 340 μ L 5mg/mL Ru (bpy)
3 2+Aqueous solution, uniform stirring reaction 30min forms stable Water-In-Oil system under the room temperature, again adding 100 μ L TEOS and 60 μ L NH in this system
3H
2O comes initiated polymerization, continue stirring reaction 20h under the room temperature, after reaction is finished, adding 5mL acetone in the system makes nano particle precipitation and separation is out from water in oil system, then with the centrifugal 10min of 8000r/min, can see having orange-yellow precipitation to form abandoning supernatant in the centrifuge tube bottom, with absolute ethyl alcohol and water difference washing precipitation several, fully remove the Ru (bpy) of surfactant TEOS and nano grain surface absorption
3 2+, get SiO
2@Ru microballoon; The preparation of nano immunity marker: AFP antibody modification SiO
2@Ru microballoon: 2mL SiO
2@Ru is diluted to 6mL with ethanol, adds 400 μ L APTS stirring reaction 30min, and is centrifugal, and water and ethanol washing obtain the SiO that APTS modifies for several times to remove unnecessary APTS
2@Ru; SiO with the APTS modification
2The glutaraldehyde solution of@Ru nano particle and 5mL 5wt% is at 37 ℃ of stirred in water bath reaction 2h, and is centrifugal and be scattered in the 3mL distilled water, and adds 2mL anti-AFP (Ab2), again at 37 ℃ of stirred in water bath reaction 2h, the centrifugal Ab2-SiO that obtains
2@Ru; At last, with Ab2-SiO
2@Ru is scattered in 5mL 1wt%BSA solution and stirs 30min, seals unnecessary amino, and is centrifugal, and with Ab2-SiO
2@Ru is dispersed to 2mL 0.02MpH 7.0PBS, and 4 ℃ save backup, and can obtain electrochemiluminescimmunosensor immunosensor thus.
Embodiment 2:
The preparation of Ab1-MUA/MU-Au modified electrode
The spun gold electrode boils 2h in 2M KOH, clean.Put it into 30 μ L 2.5mM MUA and 30 μ L 7.5mM MU (V
MUA/ V
MU=1: 10h. obtains the spun gold electrode of MUA/MU modification and immerses TNTU (o-(5-norbornene-2 in the mixed solution 3), 3-dicarboximido)-N, N, N ', N '-tetramethyluronium tetrafluoroborate) activates 15min in the activated solution, 100 μ Lanti-AFP solution 1h are put in drip washing.Put into 100 μ L BSA (1wt%) solution 30min after the drip washing with the sealing nonspecific binding site, obtain the spun gold electrode (Ab1-MUA/MU-Au) that anti-AFP (Ab1) modifies.
AFP antibody modification Si02@Ru microballoon
2mL SiO
2@Ru is diluted to 6mL with ethanol, adds 400 μ L APTS stirring reaction 30min, and is centrifugal, and water and ethanol washing obtain the SiO that APTS modifies for several times to remove remaining APTS
2@Ru.Above-mentioned nano particle and 5mL glutaraldehyde solution (5wt%) are reacted 2h 37 ℃ of stirred in water bath.Centrifugal and be scattered in the 3mL intermediate water, and add 2mL anti-AFP (Ab2), again at 37 ℃ of stirred in water bath reaction 2h, centrifugally obtain the SiO that Ab2 modifies
2@Ru (Ab2-SiO
2@Ru).At last, with Ab2-SiO
2@Ru is scattered in 5mL 1wt%BSA solution and stirs 30min, seals unnecessary amino.Centrifugal, and with Ab2-SiO
2@Ru is dispersed to 2mL 0.02M pH 7.0PBS, and 4 ℃ save backup.
The electrogenerated chemiluminescence sandwich immunoassay detects
Ab1-MUA/MU-Au inserts 100 μ L and contains in the solution of different AFP concentration, incubation 30min in 37 ℃ of water-baths.Obtain Ag-Ab1-MUA/MU-Au by first step immune response.Bathed 30 minutes with bovine serum albumin(BSA) (BSA) the solution temperature of 100 μ L1wt% again, insert 0.5mL Ab2-SiO after the drip washing
2@Ru stirs 30min to catch Ab2-SiO
2@Ru because the AFP second antibody is contained on the mark surface, therefore, can again by the combination of antigen-antibody, can modify electrode surface with this mark.Fully drip washing obtains Ab2-SiO
2@Ru-Ag-Ab1-MUA/MU-Au.
Three electrode work systems: the spun gold electrode is working electrode, and platinum electrode is to electrode, and contrast electrode is Ag/AgCl electrode (saturated KCl).Detection cell will be over against photomultiplier (PMT), and buffer solution is that 2mL contains 3m mol/L C
2O
4 2-0.1mol/L PBS (pH7.0).Photomultiplier high pressure 1000V, static lower cyclic voltammetric (CV) scanning, the synchronous recording ECL signal of in the 0.6-1.25V potential region, carrying out.
Embodiment 3:
Electrochemiluminescdetection detection:
1) optimization of test condition:
A) C
2O
4 2-As important reactant, its concentration is very large on the impact of ECL.In the constant situation of other conditions, the ECL signal value is along with C
2O
4 2-The increase of concentration raises before this gradually, after reach a platform.At C
2O
4 2-When concentration was less, the ECL signal magnitude mainly was controlled by C
2O
4 2-Concentration is therefore along with C
2O
4 2-The increase of concentration and proportional increase; Work as C
2O
4 2-When concentration increased to a certain degree, the ECL signal magnitude mainly was controlled by SiO constant in the optimization system
2@Ru concentration, thereby platform appears.Final optimization pass C
2O
4 2-Concentration is 3mmol/L.
B) antigen incubative time: along with the growth of incubative time, the ECL signal strengthens, and reaches platform after incubative time is 30min, and the final optimization pass incubative time is 30min.
2) Specification Curve of Increasing:
A) with same spun gold electrode incubation variable concentrations AFP standard solution, measure the ECL signal, the drawing standard curve is determined the optimum range of linearity;
B) with different spun gold electrodes, incubation variable concentrations blood AFP standard solution is measured the ECL signal, the drawing standard curve.
Claims (1)
1. the preparation method of an electrochemiluminescimmunosensor immunosensor is characterized in that comprising being prepared as follows step:
(1) SiO
2The preparation of@Ru microballoon: with the 7.5mL cyclohexane, 1.8mL n-hexyl alcohol and 1.77mLTween-80 mix, and then add 340 μ L5mg/mL Ru (bpy)
3 2+Aqueous solution, uniform stirring reaction 30min forms stable Water-In-Oil system under the room temperature, again adding 100 μ LTEOS and 60 μ LNH in this system
3H
2O comes initiated polymerization, continue stirring reaction 20h under the room temperature, after reaction is finished, adding 5mL acetone in the system makes nano particle precipitation and separation is out from water in oil system, then with the centrifugal 10min of 8000r/min, can see having orange-yellow precipitation to form abandoning supernatant in the centrifuge tube bottom, with absolute ethyl alcohol and water difference washing precipitation several, fully remove the Ru (bpy) of surfactant TEOS and nano grain surface absorption
3 2+, get SiO
2@Ru microballoon;
(2) preparation of nano immunity marker: AFP antibody modification SiO
2@Ru microballoon: 2mL SiO
2@Ru is diluted to 6mL with ethanol, adds 400 μ LAPTS stirring reaction 30min, and is centrifugal, and water and ethanol washing obtain the SiO that APTS modifies for several times to remove unnecessary APTS
2@Ru; SiO with the APTS modification
2The glutaraldehyde solution of@Ru nano particle and 5mL 5wt% is at 37 ℃ of stirred in water bath reaction 2h, and is centrifugal and be scattered in the 3mL distilled water, and adds the antibody of 2mL anti-AFP, again at 37 ℃ of stirred in water bath reaction 2h, the centrifugal Ab2-SiO that obtains
2@Ru; At last, with Ab2-SiO
2@Ru is scattered in 5mL 1wt%BSA solution and stirs 30min, seals unnecessary amino, and is centrifugal, and with Ab2-SiO
2@Ru is dispersed to 2mL 0.02M pH 7.0PBS, and 4 ℃ save backup, and can obtain electrochemiluminescimmunosensor immunosensor thus.
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| CN102520168B (en) * | 2011-11-25 | 2014-05-14 | 江南大学 | Immunosensor for detecting aspergillus parasiticus used for producing aflatoxin and preparing method thereof |
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| CN102778453B (en) * | 2012-08-08 | 2013-10-09 | 济南大学 | Manufacture method and application of silver hybridization SBA-15 electrochemical luminescence immunosensor |
| CN103940878B (en) * | 2014-05-05 | 2016-03-30 | 福州大学 | A kind of without reagent electrochemical luminous sensor and preparation method thereof |
| CN104865242B (en) * | 2015-03-02 | 2016-08-24 | 济南大学 | A kind of Aflatrem built based on NPCo/Co3O4-Au/RuSi@Ru (bpy) 32+ and the preparation method and application of steroids Electrochemiluminescsensor sensor |
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| CN106066324B (en) * | 2016-05-30 | 2017-08-15 | 济南大学 | A kind of preparation method of electroluminescent chemiluminescence biosensor label |
| CN110018144B (en) * | 2019-04-17 | 2021-11-09 | 南京理工大学 | Inorganic ion electrochemiluminescence detection method based on ZnTCPP @ MOF |
| CN110530853B (en) * | 2019-09-16 | 2021-09-21 | 南京工业大学 | Method for detecting aflatoxin B1 based on visual BPE-ECL technology |
| CN111220672B (en) * | 2020-02-15 | 2022-03-18 | 江苏大学 | Detection of Hg based on energy resonance transfer2+Preparation method of self-enhanced electrochemiluminescence aptamer sensor |
| CN112694394B (en) * | 2020-08-25 | 2022-05-27 | 天津大学 | Ultrasensitive multi-output signal biosensor and preparation method and application thereof |
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